The neural crest is a transient embryonic precursor population, whose derivatives include cells having widely different morphologies, characteristics and functions. These derivatives include the neurons and glia of the entire peripheral nervous system, melanocytes, cartilage and connective tissue of the head and neck, stroma of various secretory glands and cells in the outflow tract of the heart (for review, see Anderson, D. J. (1989) Neuron 3:1-12). Much of the knowledge of the developmental potential and fate of neural crest cells comes from studies in avian systems. Fate maps have been established in aves and provide evidence that several different crest cell derivatives may originate from the same position along the neural tube (Le Dourain, N. M. (1980) Nature 286:663-669). Schwann cells, melanocytes and sensory and sympathetic neurons can all derive from the truncal region of the neural tube. On the other hand, some derivatives were found to originate from specific regions of the crest, e.g., enteric ganglia from the vagal and sacral regions. These studies also revealed that the developmental potential of the neural crest population at a given location along the neural tube is greater than its developmental fate. This suggests that the new environment encountered by the migrating crest cells influences their developmental fate.
Single-cell lineage analysis in vivo, as well as clonal analysis in vitro, have reportedly shown that early avian neural crest cells are multipotential during, or shortly after, their detachment and migration from the neural tube. In avian systems, certain clones derived from single neural crest cells in culture were reported to contain both catecholaminergic and pigmented cells (Sieber-Blum, M. et al. (1980) Dev. Biol. 80:96-106). Baroffio, A. et al. (1988) Proc. Natl. Acad. Sci. USA 85:5325-5329, reported that avian neural crest cells from the cephalic region could generate clones which gave rise to highly heterogeneous progeny when grown on growth-arrested fibroblast feeder cell layers.
In vivo demonstration of the multipotency of early neural crest cells was reported in chickens by Bronner-Fraser, M. et al. (1989) Neuron 3:755-766. Individual neural crest cells, prior to their migration from the neural tube, were injected with a fluorescent dye.
After 48 hours, the clonal progeny of injected cells were found to reside in many or all of the locations to which neural crest cells migrate, including sensory and sympathetic ganglia, peripheral motor nerves and the skin. Phenotypic analysis of the labelled cells revealed that at least some neural crest cells are multipotent in vivo.
Following migration from the neural tube, these early multipotent crest cells become segregated into different sublineages, which generate restricted subsets of differentiated derivatives. The mechanisms whereby neural crest cells become restricted to the various sublineages are poorly understood. The fate of neural crest derivatives is known to be controlled in some way by the embryonic location in which their precursors come to reside (Le Douarin, N. M. (1982) The Neural Crest., Cambridge University Press, Cambridge, UK). The mechanism of specification for neural crest cells derivatives is not known. In culture studies described above, investigators reported that clones derived from primary neural crest cells exhibited a mixture of phenotypes (Sieber-Blum, M. et al. (1980) ibid; Baroffio, A. et al. (1988) ibid; Cohen, A. M. et al. (1975) Dev. Biol. 46:262-280; Dupin, E. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1119-1123). Some clones contained only one differentiated cell type whereas other clones contained many or all of the assayable crest phenotypes.
The observation that apparently committed progenitors and multipotent cells coexist in the neural crest may be interpreted to reflect a pre-existing heterogeneity in the population of primary crest cells or it may reflect asynchrony in a population of cells that undergoes a progressive restriction in developmental potential. Given the uncertainty in the art concerning the developmental potential of neural crest cells, it is apparent that a need exists for the isolation of neural crest cells in clonal cultures. Although culture systems have been established which allow the growth and differentiation of isolated avian neural crest cells thereby permitting phenotypic identification of their progeny, culture conditions which allow the self-renewal of multipotent mammalian neural crest cells have not been reported. Such culture conditions are essential for the isolation of mammalian neural crest stem cells. Such stem cells are necessary in order to understand how multipotent neural crest cells become restricted to the various neural crest derivatives. In particular, culture conditions which allow the growth and self-renewal of mammalian neural crest stem cells are desirable so that the particulars of the development of these mammalian stem cells may be ascertained. This is desirable because a number of tumors of neural crest derivatives exist in mammals, particularly humans. Knowledge of mammalian neural crest stem cell development is therefore needed to understand these disorders in humans. Additionally, the ability to isolate and grow mammalian neural crest stem cells in vitro allows for the possibility of using said stem cells to treat peripheral neurological disorders in mammals, particularly humans.
The neuregulin family of ligands are the products of a gene whose mRNA transcripts are alternatively spliced to generate a complex family of ligands. This family, recently termed "neuregulins" (Marchionni et al., Nature 362:312-318 (1993), hereby expressly incorporated by reference), was independently discovered in several laboratories. Members of this family include glial growth factor and heregulin in humans, (Holmes et al., Science 256:1205-1210 (1992), neu differentiation factor (NDF) in rats (Wen et al., Cell 69:559-572 (1992)), and acetylcholine receptor inducing activity (ARIA) in chick (Falls et al., Cell 72:801-815 (1993).
These ligands appear to bind at least two receptors, erbB2/HER2/c-neu, also called p185.sup.erbB2 (Peles et al., Cell 69:205-216 (1992)) and erbB4/HER4, also called p180.sup.erbB4 (Plowman et al., Nature 366:473-475 (1993)). In addition, a third receptor, the erbB3 receptor, has been isolated and cloned on the basis of homology with the erbB receptor family (Kraus et al., Proc. Natl. Acad. Sci. USA 86:9193-9197 (1989)).
Glial growth factor (GGF) was originally isolated from bovine pituitary as a Schwann cell mitogen (Lemke et al. (1984) J. Neurosci. 4:75-83). Subsequently, this factor was molecularly cloned and shown to encode a family of alternatively-spliced factors that are members of the TGF-alpha superfamily of growth factors (Marchionni et al. (1992) Soc. Neuroci. Abstr. 18:392). Highly purified preparations of GGF show that there are at least three separate GGFs, termed GGF-1, similar to the Lemke et al. material, supra, GGF-II and GGF-III (Goodearl et al., J. Biol. Chem. 268(24):18095-18102 (1993)). These three GGFs have been sequenced and shown to be the products of an alternatively-spliced mRNA (Marchionni et al., (1993) Id.).
The same gene was independently cloned by its ability to encode ligands of the receptor tyrosine kinase c-neu (Holmes et al. (1992) Science 256:1205-1210; Wen et al. (1992) Cell 69:559-572). GGF2 represents one of the splice variants that can be secreted from transfected cells (Marchionni et al. (1993) Id.), facilitating studies of its biological effects. Other splice variants encode transmembrane proteins (Holmes et al. (1992) Id.; Wen et al. (1992) Id.). Taken together, these data indicate that c-neu comprises at least part of the receptor for GGF.
It is an object of the invention to provide methods utilizing neuregulins to preferentially differentiate neural stem cells to glial cells.